Re: [ccp4bb] Low resolution chain tracing

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: refmac and maps in ccp4i
From: Matthew Wilce Matthew {- dot -} Wilce {- at -} MED {- dot -} MONASH {- dot -} EDU {- dot -} AU
Date: 2007-06-17

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Subject: Re: CCP4 - Monomer library sketecher
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2007-06-18

Subject: Re: Low resolution chain tracing
From: Clemens Vonrhein vonrhein {- at -} GLOBALPHASING {- dot -} COM
Date: 2007-06-18

Hi,

you might be able to distinguish the disulfide bonds from normal
backbone when looking at the log-likelihood residual maps in SHARP:
even for data collected at about 1A we often see anomalous peaks for
sulfurs (and the data doesn't even have to be very good). And a S-S
should be fairly strong.

If you want to improve those residual maps: you can easily include a
partial model (e.g. poly-ALA backbone) into the heavy-atom refinement
and phasing in SHARP. It often clears up the residual maps (to see
your S-S and maybe Cys/met residues).

You can also include the partial model during density modification
(which uses SOLOMON) in SHARP/autoSHARP: this can help defining the
first solvent envelope much better and giving you clearer density.

If you have any slightly homologous model in the PDB: try phased
molecular replacement (e.g. with MOLREP) to place this model. Looking
at something with at least a similar fold might help you getting more
confident with your directionality.

Another thing you might want to try when looking at maps is the
B-factor sharpening (see
e.g. http://atb.slac.stanford.edu/public/papers.php?sendfile=194 for a
paper). It is fairly trivial to use CCP4 programs to get the optimal
B-factor according to that paper (let me know if you need help with
this).

Cheers

Clemens

On Fri, Jun 15, 2007 at 10:03:32AM -0700, Alexei Datsuk wrote:
> Hi,
>
> I need some advice from experts. I have a low resolution electron density
> map (3.7 angstroms) solved by SIRAS. The protein is a tetramer (47
> kDa/monomer) but I'm having trouble getting in register. There is very good
> main chain density and clear secondary structure (beta-sheets) so I likely
> have solved it correctly. I can trace most of the main chain as fragments
> (loops connecting beta strands are broken) but I can't tell if I'm tracing
> it in the right direction or whether I have it backwards. There is no real
> density for side chains at all (except for Calpha), not even aromatics, so I
> have no clue which way is N or C-terminus. There are a few disulfide bonds
> too so I'm real worried about tracing incorrectly and never being able to
> fix it later. My strategy right now is to build in as much poly-ala
> fragments (I can get ~75% of trace as fragments) and then refine them. Then
> redo phasing in SHARP with poly-ala model and SIRAS phases and then use DM
> with NCS averaging to get improved map. Hopefully I can see then side chain
> density. My question is how much bias would be incorporated if some of the
> fragments are traced backwards? Would I be able to sort things out if some
> fragments are traced backwards? Has anyone traced chains as fragments not
> knowing which direction is which and then be able to sort things out later?
> Right now I am stuck- I have spent the last 1 months trying to get things in
> register but no luck. I don't want to build haphazardly but it doesn't look
> like I have much choice.
>
> What have people done when tracing in low resolution maps?
>
> Thanks
>
> Alexei

--

***************************************************************
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
* Global Phasing Ltd.
* Sheraton House, Castle Park
* Cambridge CB3 0AX, UK
*--------------------------------------------------------------
* BUSTER Development Group (http://www.globalphasing.com)
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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: refmac and maps in ccp4i
From: Matthew Wilce Matthew {- dot -} Wilce {- at -} MED {- dot -} MONASH {- dot -} EDU {- dot -} AU
Date: 2007-06-17

Next message:
Subject: Re: CCP4 - Monomer library sketecher
From: Eleanor Dodson ccp4 {- at -} YSBL {- dot -} YORK {- dot -} AC {- dot -} UK
Date: 2007-06-18